Sulfur isotope analysis by MC-ICP-MS and application to small medical samples

We describe a technique of S isotope analysis in sulfate form with the first separation stage involving anion-exchange and the second stage of mass-spectrometric analysis by MC-ICP-MS using standard-sample-standard bracketing. Ammonium in 1 : 1 stoichiometric proportion with sulfate was used to improve transmission and stability and to avoid cone and membrane clogging by condensable species. The working resolution of similar to 9000 allowed the main interferences, notably (SH)-S-32 on S-33, to be resolved. The matrix effect caused by phosphorus present in biological samples is negligible for S/P ratios \textgreater= 10: our chemical protocol allows S/P \textgreater= 150 to be routinely achieved. Replicate measurements of S standard solutions give values of isotopic abundances within errors of accepted values and demonstrate a reproducibility of +/- 0.10 parts per thousand for delta S-34 and +/- 0.15 parts per thousand for delta S-33 (2s). The technique is adequate for quantities as small as 10 nanomoles. We investigated the delta S-34 of 110 samples of cancer patients and 10 samples of rheumatoid arthritis patients. We avoided the use of blood collection tubes with sulfate-containing heparin. Sulfur in serum is transported by albumin and fibrinogen. Most serum and plasma delta S-34 values fall within a narrow interval of similar to 1 parts per thousand around a mean delta S-34(VCDT) of similar to 6.0 parts per thousand. The delta S-34 values of total blood, serum, and plasma are very similar. Despite the short turnover time of albumin and fibrinogen, S is surprisingly well regulated. Subtle variations of 0.2-0.3 parts per thousand around the mean value can be assigned to sex and age, with sulfur in male and adult samples tending to be heavier than in their female and juvenile counterparts. This narrow range of variations across the spectrum of a large number of individuals not selected for controlled dietary habits seems paradoxical. In general, breast and prostate cancer and rheumatoid arthritis have very little effect on the average serum delta S-34, but increase the scatter of values. We confirm that the serum of patients affected by liver cancer and other pathologies is depleted of albumin-born sulfur. While sulfur in the serum of patients with non-malignant liver pathologies tends to be isotopically light, the serum delta S-34 of medicated hepatocellular carcinoma patients tends to be at the high end of control values

Blood samples collection protocol for STEMI cohort at our institution.

<p>Blood samples collection protocol for STEMI cohort at our institution.</p

In-hospital management.

<p>In-hospital management.</p

Leucocytes and neutrophil granulocytes at 24 hours (H24) and their correlation with myocardial infarct size (IS).

<p><b>A</b>; Leucocytes count at H24 was significantly correlated with IS as measured by peak troponin release. <b>B</b>; At H24 neutrophil count was available for 24 patients only. For these patients, we found a significant correlation between neutrophil count and IS as measured by peak troponin release. Dotted line shows 95% confidence bands.</p

IL-17A activity assessed by ΔIL-8 on Human Umbilical Vein Endothelial Cells (HUVEC) and its correlation with infarct size (IS) in STEMI patients.

<p><b>A, B and C</b>; IL-17A activity (ΔIL-8) at H0 was not correlated with IS as measured by peak troponin level (A) (r = 0.2576, p = 0.27) peak CK level (B) (r = 0.1753, p = 0.45) or Cardiac Magnetic Resonance (CMR) (C) (r = -0.09123, p = 0.71). <b>D, E and F</b>; ΔIL-8 at H4 was not correlated with IS as measured by peak troponin level (D) (r = 0.03628, p = 0.88) peak CK level (E) (r = 0.2137, p = 0.36) or CMR (F) (r = -0.1615, p = 0.50). Correlations were tested using Spearman correlation. CK: Creatine Kinase.</p

Principal biological characteristics of the study population at admission and myocardial biomarkers peak values.

<p>Principal biological characteristics of the study population at admission and myocardial biomarkers peak values.</p

IL-17A functional test with Human Umbilical Vein Endothelial Cells (HUVEC).

<p><b>A</b>; HUVECs were incubated 48 hours with the serum of each patient. IL-8 production by HUVECs was significantly higher in STEMI patient (at H0 and at H4) compared to healthy control. <b>B</b>; HUVECs were also incubated with each patient serum in the presence of anti-IL-17A antibody (neutralizing antibody). The difference between IL-8 secretion by HUVECs without and with IL-17A neutralizing antibody (named ΔIL-8) represented the secretion of IL-8 due to IL-17A. ΔIL-8 was significantly increased at H0 for STEMI patients compare to healthy controls but not at H4. STEMI: ST-Segment Elevation Myocardial Infarction. *p<0.05, **p<0.01.</p

Early kinetics of serum Interleukine-17A and infarct size in patients with reperfused acute ST-elevated myocardial infarction

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