ANALYTICAL BIOCHEMISTRY 84, 186- 1% (1978)

A Note on the Losses of Monosaccharides, Amino Sugars, and Amino Acids from Extracts during Concentration

Procedures

RODGER DAWSON AND KENNETH MOPPER’

Institut fuer Meereskunde, Kiel, and Geologisches-Palaeontologisches Institut, Hamburg, Federal Republic of Germany

Received April 25, 1977; accepted August 12, 1977

The reproducibility of recovery of components in organic extracts may be im- proved by the addition of a small quantity of glycerin prior to concentration tech- niques such as freeze-drying or rotary evaporation to dryness. Various biologi- cal and geological samples have been analyzed for three different groups of natural hydrophilic organics using modern liquid chromatographic systems in order to examine this effect in detail.

Problems arising from concentration of natural organic components to dryness were encountered during the study of monosaccharides in marine sediments and seawater (l), where it was noticed that losses occurred upon rotary evaporation of hydrolysate extracts to dryness under reduced pres- sure (<40”(Z). The ratios of recovered sugars were significantly different from those of samples which were not allowed to be concentrated to dry- ness. The losses were hypothesized to occur as a result of wall-induced condensation reactions with other hydrophilic co-extracted components (e.g., amino compounds or humic substances). To prevent the sample from drying out requires constant supervision; furthermore the subsequent transfer steps and the making up of the sample extract to a final known vol- ume are made inaccurate when dealing with small volumes. Concentration of mixtures of pure standards to dryness does not necessarily reveal losses due to wall-induced condensations and thus an allowance for its effect on samples is difficult to quantify. Incomplete redissolution of a dried extract from the walls of a glass vessel can also introduce large errors when work- ing quantitatively with small volumes of solvent.

To overcome these drawbacks, the walls of the vessels may be coated with an inert high-boiling, low-freezing point hydrophilic agent which will cause no interference in the subsequent analyses.

First analyses have shown that glycerin fulfills these requirements.

’ Present address: Anal. Kemi, Chalmers University, Gothenburg, Sweden.

0003-2697/78/0841-0186$02.00/0 Copyright 0 1978 by Academic Ress. Inc. All rights of reproduction in any form reserved.

186

LOSSES OF AMINO AND SUGAR COMPOUNDS 187

EXPERIMENTAL

Hydrolysates of samples of North Sea and Black Sea sediments (200 mg), Escherichia coli bacterial cells (ca. 100 mg), and human blood (100 ~1) were analyzed for their contents of monosaccharides, amino sugars, and amino acids after concentration by freeze-drying or rotary evaporation with and without the prior addition of glycerin (100 ~1 of a 50% glycerin solution in ethanol).

After rotary evaporation or freeze-drying in a 25-ml pear-shaped flask the samples were redissolved in a known volume of either ethanol, in the case of the monosaccharides, or sodiuim citrate buffer, pH 2.2, for the amino acid and amino sugar analyses. Redissolution of the components in a few hundred microliters of buffer or ethanol was facilitated by ultrasonic agitation or by warming of the solution and swirling and proved to be con- siderably easier for the flasks coated with glycerin.

The analytical methods employed for the detection of the various groups of compounds are based on previously described methods and liquid chromat- ographic systems for sugars, amino sugars, and amino acids (1,2). The results of some of the analyses are displayed in Figs. l-3 in the form of chromatograms representing equivalent amounts analyzed under identical conditions.

RESULTS AND DISCUSSION

Marine Sediment

The hydrolyzed samples of the North Sea sediment (0.1 g of dried sur- face sediment) showed losses of the three amino sugars investigated rang- ing from 16% for glucosamine to 23% for galactosamine, with a noticeable loss of mannosamine relative to the sample freeze-dried with glycerin as shown in Fig. 1. The amino acid analysis on the same extracts showed noticeable random losses of several amino acids (Fig. 2), in some cases amounting to over 50% (e.g., in the cases or ornithine, lysine, and methio- nine). The concentrations of some amino acids, notably serine and threo- nine, appear to remain unaltered irrespective of whether glycerin has been added to the extract.

Dramatic losses of monosaccharides are demonstrated when sediment extracts are freeze-dried without the addition of glycerin. Figure 3 clearly demonstrates this effect in the case of a hydrolyzed sample of a Black Sea sediment. It can be readily seen that the sugar ratios, in particular those of the pentoses, are drastically altered in the extract without added glycerin, thus rendering quantitative interpretation meaningless. It should be noted that slightly better yields (ca. 10% higher) were obtained for the extract without added glycerin, when rotary evaporation was substituted for freeze-drying. This suggests that rotary evaporation is the milder of the two concentration methods.

188 DAWSON AND MOPPER

-

(A) ; (B)

3

I I 40 MIN

0

-

- 40 MI N

FIG. 1. Amino sugars in a sample of 200 mg of North Sea sediment after hydrolysis with 2 ml of p-toluenesulfonic (1.8 M) under N, for 22 hr at 110°C. (A) Without and (B) with the addition of 50 ~1 of glycerin. Vertical scale reads in absorption units; GLU NH,, GAL NH,, and MAN NH, are glucosamine, galactosamine, and mannosamine respectively.

Biological Samples

In contrast to sediment extracts the addition of glycerin to the hydroly- sates ofE. coli bacterial cells walls and human blood showed no detectable increase in the yields of amino acids or amino sugars when the extracts were not cleaned up by ion-exchange procedures. The presence of con- siderable amounts of fatty residues in these extracts may perform a function similar to that of the added glycerin, i.e., coat the walls of the vessel and prevent wall-activated condensation reactions from taking place. How- ever, when these extracts were cleaned up or desalted on cation-exchange resin, small losses (ca. 10%) of the components were detected in the case of the samples dried without the addition of glycerin. An explanation of this

LOSSES OF AMINO AND SUGAR COMPOUNDS 189

TIME

FIG. 2. Amino acid analyses of the same sample of 200 mg of North Sea sediment without (A) and with (B) the addition ofglycerin to the extract before drying. Sample was hydrolyzed with 2 ml of 6 N HCI for 22 hr at 110°C under N, before being divided into two equal parts.

phenomenom may he in the fact that lipids released from the extracts are removed during cleanup steps. Similarly, sugar analyses performed on the samples of E. co/i bacterial cells (which visibly contained fatty material in the final extract) showed little difference in the recovered amounts after drying with or without glycerin. (The desalting cleanup stage for sugar analysis involves collecting the water eluate from a mixed anion and cation exchange resin column and the lipids are partially co-eluted; the amino acids and amino sugars are cleaned up by first trapping the components onto a cation-exchange resin and then eluting with ammonia, and thus in- terfering neutral components as well as fatty compounds are to a large ex- tent washed out of the resin prior to elution of the compounds of interest.)

Some losses of glucose, galactose, and fructose were, however, noted from the sample of hydrolyzed blood cells dried without the addition of glycerin (amounting to only around lo- 15%) but may only reflect differ- ences in purity of the extract compared with the bacterial sample.

DAWSON AND MOPPER

(A) WITHOUT GLYCERIN

GUI Man Fru Xyi Am Lyx Rib Fuc Rho -.OL

9 2

-

2 (8) WITH GLYCERIN

z -.06 $

1 -.06 9

-.04

-02 -

l-0 l60 120 60 a

b-4 clime

FIG. 3. Effect of adding glycerin before freeze-drying on the sugar yields from a Black Sea sediment hydrolysate (2 ml, 2.0 N HCl, 3 hr, 110°C). (A) Without and (B) With the addition of glycerin. Glu, glucose; Gal, galactose; GUI, gulose; Man, mannose; Fru, fructose; Xyl, xy- lose; Ara, arabinose; Lyx, lyxose; Rib, ribose; Fuc, fucose; and Rha, rhamnose.

CONCLUSIONS

It is a common practice to purify a sample as far as possible before in- jection into a sensitive analyzer and this procedure may remove “internal protective agents” in the form of viscous liquids which coat the walls of glass vessels during evaporation to dryness, prevent condensation reac- tions, and aid the redissolution of the sample prior to analysis. Unless a coating agent is artificially added, both quantitative and qualitative analy- sis may be rendered meaningless by random losses of natural compounds.

In reports dealing with the analysis of sugars and amino acids in seawater and sediments (3) the reliability of past data has been questioned, since the methodologies employed took little or no account of this phenomenom.

ACKNOWLEDGMENTS

The authors wish to thank Mrs. B. Lohmann for assistance in preparing the samples for analysis and Dr. K. Gocke for providing the bacterial cultures. We are grateful for the valuable discussions with Professor E. T. Degens and Dr. C. Garrasi. The research was jointly sup- ported by the Sonderforschungsbereich and the German Research Society.

REFERENCES

I. Mopper, K. (1973) Ph.D. Thesis, Mass. Institute of Technology-Wood’s Hole Oceanographic Institute.

2. Mopper, K., and Degens, E. T. (1972)AnaL Biochem. 45,147; Mopper, K., and Gindler, E. M. (1973) Anal. Biochem. 56, 440; Dawson, R., and Pritchard, R. G. (1977) Mar. C/rem., 5, in press. Dawson, R., and Mopper, K. (1977) in preparation.

3. Dawson, R., and Pritchard, R. G. (1977) Mm. Chem.. 5, in press.